Acute functional tolerance (AFT) to ethanol is a well known phenomenon that is thought to influence individual differences in risk liability for alcoholism, but the underlying molecular substrates of AFT remain a mystery. In this application, we are proposing to test the hypothesis that the zebrafish (Danio rerio) will exhibit AFT to the behavioral disruptions produced by ethanol, with the main goal of the proposal to characterize and develop the zebrafish as a model organism in the study of the molecular basis of AFT. Zebrafish provide numerous advantages for genetic studies including short generation time, large numbers of offspring, low cost and space demands, a growing body of information on the genetics of zebrafish, and the ease with which genetic manipulations can be accomplished. Another important advantage of zebrafish in AFT studies is that a steady-state blood and brain ethanol concentration can easily be maintained since the ethanol is administered via equilibration with the holding tank solution; in essence, an extremely simple ethanol clamp. AFT for certain behaviors has been demonstrated in goldfish, but it has not been examined in zebrafish. The Specific Aims of this project are to screen several established zebrafish behaviors for the acquisition of AFT, determine if there is naturally occurring genetic variation for AFT by testing several inbred zebrafish strains, and to develop methods for automated, high-throughput mutagenesis screens for AFT. Future studies will implement the high-throughput screens on large numbers of mutagenized zebrafish with the goal of mapping the molecular basis of AFT in zebrafish.